The contribution of reticulocytes and other young RBC toward both the hemolytic and vasocclusive aspects of sickle cells disease has become more apparent in recent years. At the same time, effective treatment strategies (hydroxyurea) and promising experimental agents (clotrimazole) have become available. These agents induce fundamental but poorly understood changes in the behavior of sickle cells. In the proposed research, studies will be performed to better understand the time-dependent cellular changes that occur in circulating sickle cells, the role of cell age and HbF content in determining these changes, and the modification of sickle cell behavior that occurs as a result of effective treatment. Three types of experiments will be performed: 1) Detailed in vitro analysis of RBC subpopulations in density fractions. These experiments will shed light on the extent of dehydration as a function of age and HbF content, and on changes in the behavior of these cellular subtypes with treatment; 2) Investigations of the transport pathways that lead to dehydration of young and mature RBC under oxy and deoxy conditions, and the changes in the activity of these pathways with treatment; 3) In vivo, multiparametric tracking of biotin- labeled, autologous sickle cells to determine the survival and time- dependent hydration change of RBC subtypes, the influence of cell age and HbF, and the changes that occur after effective treatment. The observed red cell behavior, before and after treatment, will be analyzed in the context of a comprehensive model of sickle cell dehydration and survival. In this model, initial reticulocyte dehydration is dependent on the activity of the CI co-transport pathway for K efflux and is independent of deoxygenation and HbF, while terminal stages of dehydration are sickling dependent, with HbF playing an important role. These investigations will provide a better understanding of sickle cell pathophysiology, define the cellular changes that occur as a result of effective treatment, and provide the information necessary to formulate improved treatment strategies.